FVIII is a large multi-domain protein of 2,332 amino acids made up of three structural domains, A, B and C which are arranged in the order A1:a1:A2:a2:B:a3:A3:C1:C2. The A domains possess more than 40% homology and are also homologous to ceruloplasmin (for recent review, see Pratt (2000) and Saenko (1999)). 30% homology also exists between the A domains of factor V and FVIII. The C domain occurs twice and is reported to be able to bind glyco-conjugates and phospholipids having a net negative charge. It exhibits homology with lectins which are able to bind to negatively charged phospholipids. The platelet attachment site has been located in this region (C2 domain) (Foster et al., (1990)).
These antigenic determinants consist of fragments 351-365 (A1 domain—heavy chain), 713-740 (A2 domain), 1670-1684 (A3 domain—light chain) (NH2 end of the light chain) or else 2303-2332 (C2 domain—light chain) (Foster C, (1990)), fragments 701-750, 1663-1689, 330-472, 1694-1782 (EP-0 202 853), 322-740 and 2170-2322.
The U.S. Pat. No. 5,744,446 describes a hybrid human/animal Factor VIII having a sequence of amino acids selected from the group of the A2 domain fragments 373-540, 373-508, 445-508, 484-508, 404-508, 489-508 and 484-489, with corresponding sequences of porcine or murine Factor VIII, said hybrid being used for the treatment of Factor VIII deficiencies.
The antibodies which recognize these various sites interfere, with the activation of FVIII, the binding of vWf, FIXa, FXa, APC or phospholipids. The specific antibody response to FVIII varies considerably among individuals, and epitopes for inhibitor antibodies have to be determined for all FVIII domains (see for recent review Scandella, 2000; Lollar, 2000).
Other antibodies, which do not inhibit standard activity tests in vitro, can exert an influence on the behavior of FVIII with the other constituents of the coagulation cascade while attaching themselves to sites in the molecule which are at a substantial distance from the active sites. These antibodies can interfere with the natural state of folding of FVIII by altering some of its properties.
Emergence of alloantibodies (inhibitors) that neutralize infused FVIII activity may seriously complicate FVIII replacement therapy. Reported inhibitor incidence rates in hemophiliacs vary considerably. They range around 6-35% (Vermylen et al, 1998). Candidates for genetic predispositions such as large deletions and intron 22 inversion have been found associated with a high incidence of inhibitors and genes that are involved in the immune response as genes MHC class I and class II (Tuddenham and McVey, 1998). Repeat switching from one FVIII product to another and the possibility that some FVIII concentrates are more immunogenic may also explain the appearance of inhibitors (Vermylen et al, 1998). Different methods of preparing FVIII could exert an influence on its structure, its physicochemical properties or its natural microenvironment; Laub et al. (1999); Raut et al. (1998)). Clinically relevant anti-FVIII autoantibodies are rare in non-hemophilic patients (annual frequency in the population: 1-5/106) (Morrisson and Ludlam) (1995). They are associated with a number of autoimmune diseases and are often characterized by life-threatening hemorrhage. On the other hand, anti-FVIII antibodies have also been described in healthy subjects (Algiman et al, 1992; Moreau et al, 2000), without any apparent effect on the subjects' levels of circulating FVIII.
Self proteins or derived peptides may elicit an immune response if presented to CD4 T cells at inflammatory sites by professional antigen presenting cells. Using pools of overlapping synthetic peptides spanning the sequences of individual FVIII domains, Reding et al. (2000) showed reactive CD4+ to FVIII in healthy subjects and hemophilia patients. Several FVIII domains were recognized: A3 domain was recognized more strongly and frequently and each domain forms several epitopes.
Techniques such as western blotting, immunoprecipitation, and enzyme-linked immunosorbent assays (ELISAs), using well-defined FVIII proteolytic fragments, a large recombinant peptide library, or synthetic peptide arrays, have been used to map different FVIII-inhibitor binding sites located mainly in the A2 and C2 domains. However, none of these techniques has made it possible to build a model for identification of inhibitor and non-inhibitor epitopes. Only a few epitopes have been mapped to discrete sequences (<20 amino-acid residues). To solve this problem, Palmer et al (1997) synthesized 96 undecamer peptides (11 amino-acid residues) representing 80% of the complete residue sequence of FVIII. They succeeded in determining the epitope specificity of 9 patients' inhibitory antibodies. Other useful techniques are analysis of FVIII gene mutations and their effects on the FVIII molecule as well as phage display technology (van den Brink et al, 2000). All these methodologies, however, are time consuming, rather costly, and largely dependent on patient availability. Certain areas of the FVIII molecule may be “hot spots” containing commonly recognized clusters of inhibitor epitopes, e.g., regions in the A2 domain, A3 domain, and C2 domain. The reason for these “hot spots” in generating an inhibitor response remains poorly understood (Reisner et al, 1995).
Currently, a predominant notion among hemophilic patients, clinicians and “fractionators” is that of having available a purified FVIII which is devoid of all pathogenic plasma contaminants and secondary effects.
Different animal models could be used as hemophilia dogs, SCID mice, hemophilia mice . . . but until now, no satisfactory experimental model exists which makes it possible to forecast the immunogenicity or the immuno-modulatory effect of the FVIII preparations, or the susceptibility of the host, before they have been administered clinically.
Patients who develop an anti-FVIII immune response find themselves in a serious situation which necessitates the use of severe, aggressive and excessively expensive measures.
One of the frequently treatment, is the induction of immune tolerance by administration of very high doses of FVIII (150 IU/kg twice a day) in association or not with prothrombin complex concentrates and is assigned as “Bonn Protocol”. Treatment options are also to by-pass the FVIII inhibitor activity by use of PCC (preferably an activated PCC [APCC]) or FVIIa. Specific antibodies as consequence of the infusion of these alternative agents could be produced, impairing the treatment. As an alternative agent porcine FVIII may be used to achieve hemostasis in patients with antibodies that do not substantially cross-react with porcine FVIII before or during the treatment (Lollar, 2000).
A potential alternative approach to inhibit the production of inhibitors is blockade of the T cell/B cell collaboration mediated by through receptor ligand binding signal events (Ewenstein et al, 2000). Preliminary clinical trials were performed using a humanized mouse monoclonal antibody to human T cell CD40 ligand (CD 154).
A profitable strategy for reducing the level of inhibitors has consisted in subjecting patients to an extracorporeal circulation to enable solid-phase absorption of the total IgG.
The immunoabsorbant could be Sepharose-bound staphylococcal protein A or Sepharose-bound polyclonal sheep antibodies to total human immunoglobulin (Knobf and Derfler, 1999). The foreign proteins (protein A, sheep anti-human Ig) could leak from the column and triggered the immune system of the recipient; moreover problems could raise as sanitization (ICH Topic Q5A, Directive 92/79/EC).
The infusion of polyvalent intravenous immunoglobulins (IVIG), where appropriate combined with an immunosuppressive treatment, has been found to be relatively effective, although the reason for this effectiveness is still not fully established. Various hypotheses involving feed-back inhibition of IgG synthesis, stimulation of IgG clearance or activation of T suppressor cells have been advanced. An interesting explanation is that these commercial intravenous immunoglobulins might contain antibodies which are able to react with the variable parts (idiotypes) of the anti-FVIII antibodies and neutralize these antibodies (Dietrich et al. (1992)).
Unfortunately, none of these approaches has been found to be satisfactory in terms of safety, efficacy, efficiency and cost.
The state of the art in epitope structure prediction was limited given to the fact that non-continuous amino acid residues seem to constitute most important epitope and that the dynamics of binding is often not integrated into the epitope prediction equation making epitope structure prediction a complex four-dimensional problem (Van Regenmortel, Methods: A companion to Methods in Enzymology, 9, page 465-472, 1996).
According to the author, most of the antibodies raised against intact proteins do not react with any peptide fragment derived from the parent protein indicating that such antibodies are directed to discontinuous epitopes (conformational epitopes).
This author states also that low success rate of antigenic prediction is due to the fact that predictions concerns only continuous epitopes and it is unrealistic to reduce the complexity of epitopes that always possess conformational features to one dimensional linear peptide model.
Similarly, Palmer et al. (1997) using synthetic peptide arrays to identify novel Factor VIII inhibitor epitopes note that each patient pattern of anti-factor VIII antibody reactivity appears to be polyclonal, directed against multiple sites located within the amino and carboxyl terminus of the protein and seems to be unique for each plasma investigated (see also above).
Moreover, this author notes that it is difficult to predict the importance that any given antibody: epitope interaction may have on Factor VIII coagulation activity based on the results of synthetic peptide assays alone (due to the incomplete understanding of the relationship between structure and function of different factor VIII domains and the possibility that both inhibitor and non-inhibitory antibodies may be present in a patient's plasma.
Therefore, the documents of the state of the art do not suggest to identify antigenic linear peptides upon a macro-molecule (such as Factor VIII) and that linear epitopes could be used for the diagnostic and/or the therapy of immune disorders induced by inhibitors directed against Factor VIII.
The present invention aims to obtain antigenic polypeptide sequences of factor VIII, fragments and epitopes of these sequences, whose purpose is to improve the diagnosis and/or therapy (including prevention) of immune disorders (in particular those induced by inhibitors of FVIII and inhibitors of FVIII, especially inhibitors of the binding of the von Willebrand factor (vWf), to the FIX and/or to membrane phospholipids (PL)), and which allows a screening between non-inhibitory and inhibitory anti-FVIII allo- or auto-antibodies (allo- or auto-immunoglobulins).
Another aim of the invention is to obtain inhibitors which exhibit an immunoaffinity with these antigenic polypeptide sequences, fragments and/or epitopes, as well as to obtain anti-inhibitors, in particular antibodies or (T) cell receptors, which are directed against the above-mentioned said inhibitors and whose purpose is to improve the diagnosis and/or therapy (or prevention) of immune disorders.
A further aim of the invention is to obtain said molecules at high purity, in industrial level, without contaminants (viruses, prions, . . . ) and according to the GMP practices in the field of therapy and diagnostics (ICH topic QSA, Directive 92/79/EC, etc.).